Antibodies directed to specifically chosen antigens have been used in the treatment of various conditions. For example, Campath-1 monoclonal antibodies (mAb) have been used successfully to induce remissions in lymphoma and leukemia patients and for the treatment of rheumatoid arthritis and vasculitis. The target antigen, CD52 (also referred to as CDw52; see e.g. Xia et al., 1991), is a GPI-anchored glycoprotein of lymphocytes and monocytes (and parts of the male reproductive system). It has an exceptionally short peptide sequence of 12 amino acids and a single, complex, N-linked oligosaccharide at Asn3 (Hale et al, 1990; Xia et al, 1991). CD52 is a good target for antibody-mediated killing and is therefore an effective cell surface molecule for various therapeutic regimens in which reduction in lymphocytes is an objective (e.g. removal of cells from donor bone marrow to prevent graft-versus-host disease, treatment of leukemia and lymphoma, and immuno-suppression).
Several rat anti-human CD52 Campath-1 mAb were generated by fusion of the Y3 rat myeloma line with spleen cells from a rat immunized with human T lymphocytes (Hale et al, 1983). Although the clinical effectiveness of rat Campath-1 mAb has been demonstrated regularly, many patients mounted an anti-antibody (antiglobulin) response against the xenogeneic protein that prevented retreatment with the therapeutic antibody. Antibody therapy is often limited by the antiglobulin response. The anti-idiotypic component (anti-Id; directed against the Ab V regions and in particular the Ab-combining site) inhibits the binding of the Ab to its target while both the anti-Id and the anti-isotypic component (directed against the constant regions) act to accelerate antibody clearance. A major concern is the neutralizing effect of the antiglobulin response. As with antiglobulin responses in general, anti-Id responses interfere with the clinical potency of a therapeutic Ab by forming Ab aggregates that are rapidly cleared from the circulation, reducing the chance for interaction with target antigen. Unfortunately, most antiglobulin sera contain anti-Id antibodies. This has been demonstrated for a number of therapeutic mAb and is especially noted after repeated treatments.
To reduce the immunogenicity of the rat IgG2b Campath-1 antibody, YTH34-5, the gene fragments encoding the VL and VH were humanized by “CDR grafting” of the rodent hypervariable regions onto human framework regions (Jones et al, 1986; Reichmann et al, 1988). This was carried out by splicing the CDR sequences encoding the rat Campath-1 antibody onto sequence encoding human framework backbone provided by the crystallographically solved myeloma proteins NEW (for the VH) and REI (for the VL). The resulting protein had low antigen-binding titre and modelling of the humanized V-region showed that residue 27 in the VH framework sequence was critical for preserving the loop structure of CDR1. This residue was changed from the residue found in NEW (Ser) back to the rat residue Phe which resulted in restoration of antigen binding. During the to modelling, an additional change (NEW residue Ser to the rat residue Thr) was also suggested. However, in functional assays this substitution had no effect on antigen binding, but the double mutant (Ser27 to Phe27 and Ser30 to Thr30) expressed the most protein and therefore was used to produce therapeutic humanized Campath-1 Ab, designated Campath-1H (Reichmann et al, 1988). As many human VH frameworks have threonine at position 30, this change was not considered an additional risk to the antibody's immunogenicity. The humanized VL and VH were then genetically fused with human light chain and heavy chain constant regions, respectively. In summary, the humanized Campath-1 antibody consists of human residues at all positions except those encoding the 3 CDRs of the light chain, the 3 CDRs of the heavy chain, and residues Phe27 and Thr30 in VH of the heavy chain.
In clinical trials, the humanized version (Campath-1H) was found to be much less immunogenic than the rat IgG2b Campath-1 antibody. Humanization reduces the immunogenicity of rodent mAb, although both the idiotype and the allotype of a humanized mAb might stilt be targets for humoral responses. Sensitization to idiotype has indeed been documented in some allotype-matched recipients of Campath-1 H (Isaacs et al, 1992; Lockwood et al, 1993). These responses were revealed by the presence of anti-id in the patients' sera. One patient generated high-titre anti-Id that crossreacted on the entire panel of CD52 mAb. Humanized Campath-1 antibodies are described in EP 0 328 404 the teachings of which are incorporated herein by reference, in their entirety.
One strategy to further reduce the immunogenicity of Campath-1H might be to re-graft the 6 CDR loops onto well-characterized human germline framework regions. The majority of the humanized V regions so far have used rearranged V-genes as acceptor framework sequence. This was the case for Campath-1H as framework sequences from myeloma proteins were used to provide acceptor sequences for both VH and VL. Rearranged V-genes often contain somatic mutations, acquired during the process of affinity maturation. These will be unique to the individual from which the rearranged genes were derived and therefore may be seen as foreign in another individual. However, there is a possibility that regrafting may introduce new idiotypic epitopes, formed by the junctional regions encompassing CDR residues and new framework residues. Furthermore, humanization alone may not solve the problem of anti-Id responses because the human population is outbred and it is unlikely that all patients will be tolerant to a given humanized mAb. Even in antibody constant regions, there are a number of different alleles which carry allotypic markers to which naturally occurring antiglobulin responses can be demonstrated. The problem is more complex for V-region segments, which show a higher degree of variation both in allotype and haptotype in comparison to constant regions.
Another approach is to induce tolerance to the potentially foreign peptides contained within the Campath-1H V-region. We know that the antiglobulin response is itself a B-cell response which is CD4+ T-cell dependent. Isaacs and Waldmann (1994) demonstrated that mice deprived of CD4+ Tells were unable to respond to a foreign cell-binding mAb (rat anti-mouse CD8 mAb). CD4+ Tell depletion was carried out by adult thymectomy combined with administration of a depleting CD4 mAb. In these mice, the response to subsequently administered mAb or SRBC was measured. CD4+ T-cell deficient mice failed to make either an antiglobulin response or an anti-SRBC response, demonstrating that the anti-Ig response, like the anti-SRBC response, is classically CD4+ T-cell dependent. In order to generate T-cell help and to get the appropriate T-cell response, the adminstered Ab must be processed as a protein antigen and presented, presumably in the context of an MHC class II molecule, by a suitable antigen presenting cell. Therefore, two main strategies can be adopted to decrease the immunogenicity of a humanized V-region. (1) We can “silence” the antibody molecule itself, adopting strategies to eliminate any potential T helper epitopes, or (2) we can present all the potential T helper epitopes in a manner that induces tolerance instead of reactivity to those epitopes.
“Silencing” the Antibody Molecule:
a) In theory, we might be able to silence the antibody itself so that the immune system will not recognize foreign determinants. This would be possible if we could scan the VL and VH amino acid sequences for motifs that could bind to MHC class II molecules. If we could thus identify key residue(s) in a potential class II peptide that were not involved in antibody specificity or affinity, then it/they could be changed by site-directed mutagenesis to residue(s) that did not allow association with class II molecules. T helper peptides are not random, and any protein has only a limited number of peptides capable of binding to MHC class II molecules, and also to T-cell antigen receptors. However, this is not possible at present because class II-binding peptides are not yet characterized to a sufficient degree to be identified by scanning protein sequences. This is in part due to the heterogeneous nature of class II peptides. Naturally processed peptides isolated from MHC class II molecules are generally larger in size, variable in length and have both ragged ends at C- and N-termini in comparison to processed peptides isolated from MHC class I molecules. Whereas class derived peptides are mostly of uniform length of 8-9 amino acid residues, MHC class II-associated peptides range from 12-24 amino acids (Rudensky et al, 1991; Hunt et al, 1992; Rudensky and Janeway, 1993). Class I-derived peptides have sequence motifs with specific anchor residues in certain positions allowing their side chains to fit in the binding pockets of the peptide-binding groove, and the peptide-binding groove is closed at both ends. In contrast, class II peptides are bound in an extended conformation that projects from both ends of an “open-ended” antigen-binding groove; a prominent non-polar pocket into to which an anchoring peptide side chain fits near one end of the binding groove (Brown et al, 1993).b) Other strategies that might be adopted to “silence” the antibody if we could predict class II peptide-binding motifs. For example, one could include insertion of a protease cleavage site within any potential class II epitope to increase the chance of peptide degredation before they could be presented in the context of class II. Alternatively, insertion of motifs into a V-region such as Gly-Ala repeats may inhibit the degradation of the V-region into peptides that could associate with class II molecules. In one system, it was shown that EBNA1 Gly-Ala repeats generated a cis-acting inhibitory signal that interfered with antigen processing during MHC class I-restricted presentation such that CTL recognition was inhibited (Levitskaya et al, 1995). Although either of these approaches may hold some promise in the future, they again rely on prediction of potential MHC class II peptides from protein sequence of humanized VL and VH regions and are therefore limited by insufficient knowledge regarding consensus motifs for class II peptides.Inducing Tolerance to T Helper Epitopes
In lieu of sufficient knowledge regarding class II peptide motifs, we have turned our attention toward induction of tolerance to therapeutic antibodies. In 1986, Benjamin et al and Cobbold et al described an unexpected property of cell-binding mAb: whereas it was possible to induce tolerance to the Fc region (anti-isotype tolerance), the idiotype remained antigenic under equivalent conditions. Moreover, it was relatively easy to induce tolerance to non-cell binding mAb but cell-binding mAb were found to be very immunogenic.
Isaacs and Waldmann (1994) in a preliminary study used a non-cell-binding “mixed molecule” derivatives of a cell-binding Ab to induce tolerance to the wild-type form. The cell-binding antibody was an anti-CD8 mAb in a mouse model. The non-cell-binding derivatives were made by pairing the relevant L- and H-chains with an irrelevant H- or L-chain, respectively. The relevant H-chain paired with an irrelevant L-chain was obtained by limiting dilution cloning of the original hybridoma that was expressing a myeloma light chain (from the Y3 fusion partner), as well as the specific anti-CD8 H- and L-chains. A variant of the hybridoma that expressed the myeloma L-chain and the specific anti-CD8 H-chain but no anti-CD8 L-chain was obtained. A clone expressing the relevant L-chain only was also obtained in this manner. That clone was then fused to a hybridoma expressing an irrelevant specificity (anti-human CD3) and a variant was selected that expressed the relevant anti-CD8 L-chain with the irrelevant anti-CD3 H-chain. Because proteins are processed into peptides prior to presentation to T-cells, helper peptides from antigen-specific H- and L-chains would be “seen” by T-cells, regardless of their partner chain. However, in this case, there was no advantage in tolerance induction using non-cell-binding mixed molecule derivatives of a therapeutic mAb in vivo compared to an isotype-matched control, suggesting that in the strain of mice used, most (or all) of the helper epitopes were located within the constant region.
In practice, using these “mixed molecules” of antigen-specific and irrelevant immunoglobulin chains for human therapy would not be feasible because the irrelevant H- and L-chains would carry some helper epitopes themselves, thus complicating the ability to achieve tolerance to the relevant H- and L-chains. Nor would one expect to tolerize those B-cells which “see”. idiotypic determinants formed by the combination of the relevant H- and L-chains of the antibody.
Campath-1 is a cell-binding mAb, and an effective tolerogen for use with it, such as a non-cell-binding form of the therapeutic mAb would therefore be advantageous. The same goes for other therapeutic antibodies which have cell-binding properties, and non-cell-binding variants thereof.
The Invention
The invention therefore provides an antibody which is a modified version of a therapeutic antibody with affinity for a cell-surface antigen, said antibody having reduced affinity for the antigen compared with the therapeutic antibody as a result of a modification or modifications to the antibody molecule, wherein the antibody is capable of inducing immunological tolerance to the therapeutic antibody.
Preferably, the affinity of the antibody according to the invention for the antigen is reduced to 50% or less of the affinity of the therapeutic antibody for the antigen. More preferably, the affinity is reduced to 10% or less, or to 1% or less of the affinity of the therapeutic antibody. The affinity needs to be sufficiently reduced to allow the antibody according to the invention to act as a tolerogen with respect to the therapeutic antibody. The term “non-cell-binding variant” is used herein to refer to antibodies according to the invention, although antibodies according to the invention may still have some binding affinity for the cell surface antigen.
The ability of the antibody according to the invention to induce immunological tolerance to a therapeutic cell-binding antibody relies on the presence in the non-cell-binding antibody of at least one epitope also present in the therapeutic antibody, which induces an immune response in the intended patient.
The non-cell-binding antibody is preferably capable of tolerising to anti-idiotypic responses, at least to the V domain hypervariable regions of the therapeutic antibody and preferably also to the framework regions. Thus it is desirable that the tolerising antibody has an amino acid sequence similar to the therapeutic antibody in those regions. Preferably there is >90%, or >95% or >99% amino acid sequence identity between the variable domains of the non-cell-binding antibody and the therapeutic antibody. Most preferably the differences are restricted to any amino acid to substitution(s) required to sufficiently reduce antigen binding affinity in the non-cell-binding antibody.
Preferably also the non-cell-binding antibody is capable of inducing tolerance to the constant regions of the therapeutic antibody. Thus, it is preferred that the constant domains of the non-cell-binding antibody are similar to those of the therapeutic antibody, having for example >90% or >95% or >99% amino acid sequence identity. Most preferably, the constant domains of the non-cell-binding antibody and the therapeutic antibody are identical and are thus matched allotypically.
The invention further provides fragments of an antibody described herein, the fragments having tolerance-inducing capability. Such fragments include monovalent and divalent fragments such as Fab, Fab′ and F(ab′)2 fragments. Also included are single chain antibodies. The preferred features of such fragments are as described herein in relation to non-cell-binding antibodies according to the invention. The non-cell-binding fragments may be for use with corresponding therapeutic antibody fragments, or with therapeutic antibody molecules.
The reduced binding affinity of the non-cell-binding antibodies may be achieved in a variety of ways. In the preferred embodiment described herein, an alteration in the CDRs comprising one or two or more amino acid substitutions reduces binding affinity, Alternatively, amino acid substitutions in other parts of the antibody molecule may be used to reduce binding affinity. For example, amino acid substitutions in the framework regions are known to significantly affect binding affinity (Reichmann et al 1988). Another alternative is a monovalent form of the therapeutic antibody. Monovalent antibodies have reduced binding affinity compared to their bivalent counterparts. Monovalent forms may be for example Fab fragments, or single chain antibodies, or any other genetically engineered antibody fragments retaining a single binding site. Monovalent variants can also be produced by mutating the cysteine residue which participates in interchain (H-H) disulphide formation (e.g., cys→ser or cys→ala). The reduction in binding affinity of a monovalent antibody compared to its bivalent counterpart may be sufficient to enable tolerance induction. Preferably, the monovalent antibody is either incapable of binding Fc receptors, or incapable of binding complement component C1q, or both. Either or both of these properties can be introduced by suitable mutations (see e.g., Morgan et al., WO 94/29351, published 22 Dec. 1994 and Winter et al., EP 0 307 434 B1).
The non-cell-binding antibodies or fragments according to the invention may thus be one of a variety of types of antibodies or fragments, including genetically engineered antibodies or antibody fragments. In addition, the antibodies or fragments will generally be from a mixture of origins. For example, they may be chimeric e.g. human constant regions with rat variable domains; or humanised or CDR grafted or otherwise reshaped (see, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0 125 023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0 120 694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0 194 276 1; Winter, U.S. Pat. No. 5 225 539; Winter, European Patent No. 0 239 400 B1; Queen et al., U.S. Pat. No. 5,585,089; Queen et al., European Patent No. 0 451 216 B1; Adair et al., WO 91/09967, published 11 Jul. 1991; Adair et al., European Patent No. 0 460 167 B1; and Padlan, E. A. et al., European Patent No. 0 519 596 A1. See also, Newman, R. et al., Biotechnology, 10: 1455-1460 (1992), regarding primatized antibody, and Huston et al., U.S. Pat. No. 5,091,513; Huston et al, U.S. Pat. No. 5,132,405; Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988) regarding single chain antibodies). Campath-1H is considered humanised although it contains two amino acid substitutions in the framework regions.
Ideally, the antibody according to the invention is as close as possible to the therapeutic antibody on which it is based. Administration of such a “minimal mutant” prior to injection of the cell-binding therapeutic mAb can be used to tolerise to all T- and most B-cell epitopes in the therapeutic mAb. Classic experiments indicate that tolerance is maintained more effectively by T-cells than by B-cells. But since most B-cell responses including the anti-id response require T-cell help, even if a B-cell is responsive to a given antigen, antibody production will be determined by the state of responsiveness of the T-cells (Chiller et al, 1971). Thus, it will be preferable to use a non-cell-binding variant which contains the minimum differences needed to reduce its affinity for the cell-surface antigen sufficiently to enable it to be used as a tolerogen. By using techniques such as X-ray crystallography, computer modeling and site-directed mutagenesis, and also genetic methods such as phage display, it will be possible to design suitable non-cell-binding variants for any cell-binding therapeutic antibody.
The antibody according to the invention is preferably in biologically pure form, desirably being at least 95% (by weight) free of other biological materials.
As used herein, the term “cell-surface antigen” means an antigen which is found on, cell surfaces, but not necessarily exclusively on cell surfaces.
The term “therapeutic antibody” is used herein to refer to an antibody which may be administered to humans or animals to have a desired effect in particular in the treatment of disease. Such therapeutic antibodies will generally be monoclonal antibodies and will generally have been genetically engineered.
In another aspect the invention comprises a composition for administration to a patient comprising an antibody as described herein, together with a physiologically acceptable diluent or carrier.
In a further aspect, the invention provides a host cell or cell line which expresses an antibody as herein described and use of such a host cell or cell line for the production of such an antibody.
Additional aspects of the invention include the use of an antibody as described herein in the manufacture of medicament for the induction of tolerance, in particular tolerance to a therapeutic antibody.
Using Rational Design to Create a “Minimal Mutant”
In one embodiment for producing a non-cell-binding variant of a therapeutic mAb, amino acid residues which are involved in binding to target antigen are identified. Relative to the number of residues that comprise the VL and VH domains, those that are directly involved in interactions with antigen are small in number (Novotny et al, 1983). And although the Ab-combining site is made up of 6 hypervariable loops, 1 or 2 of those loops may dominate in that interaction. If a key residue or residues can be identified, it/they can be changed by site-directed mutagenesis to a residue that will reduce (reduce or abolish) antigen-binding. Because these residues will most likely be found within the hypervariable loop structures and not in the framework sequence supporting those loops, small changes may not significantly disrupt the overall structure of the Ab.
Model Building of Ag-binding Sites to Define Key Residues for Mutagenesis:
Because the constant regions and variable regions of Ab molecules are very similar in sequences and structures, general principles regarding Ab structure have been defined using relatively few solved crystal structures. To date, approximately 50 structures of Ab fragments have been included in the Brookhaven Protein Data Bank, and of these, 20% have been refined to a resolution of 2.0 angstroms or better. As the structural knowledge base increases, comparative Ab modelling (modelling by homology) becomes more reliable since there is a greater choice of structural templates. Variable regions (VL and VH) of different Ab structures can be combined as a structural template after superimposing their most conserved residues. Side chain conformations of buried residues are then modelled. The CDR loops are modelled by identification of structurally similar loop templates (often loops with the same length and similar sequence have similar backbone conformations). These CDR sequences often fall into canonical loop motifs (excluding H3, between 50 and 95% of murine VL (kappa) and VH have loop sequences consistent with classified canonical motifs). Canonical loop backbones can then be spliced onto the model of the framework and CDR side-chain conformations can be modelled based on conformations of residues found at corresponding positions in other loops of the same canonical structure. Finally the model is often refined using computer programs that minimize troublesome stereochemical constraints.
Comparative model building is becoming widely used as the size of the structural database increases, providing a greater range of structural templates. Also, the greater range of computer programs available ensures that models are becoming increasingly accurate. For example, the solved crystal structure of Campath-1H was very close to the structure predicted by molecular modelling. We could predict from the to modelling data that mutations 1 and 2 described in the Examples were likely to have a detrimental effect on binding to CD52. The crystal structure confirmed these predictions and also predicted that mutation 3 could disrupt binding to CD52.
Obviously, to create a non-cell-binding version of a therapeutic Ab, it is desirable to start with a solved crystal structure, preferably co-crystallized with antigen so that the key contact residue(s) can be identified and substituted for residue(s) that destroy antigen binding. However, in many cases, a good molecular model could provide the necessary information. In cases where the molecular model is of poor quality (for example, if the appropriate structural templates do not exist in the databank), CDR swapping experiments (as described in the Examples) will provide information on which CDRs must be targeted for mutation. Alanine scanning mutagenesis (mutating each residue sequentially to Ala) through those regions could identify the key residue(s) involved in antigen-binding (Cunningham and Wells, 1989). If changing a single residue to Ala reduced but did not destroy binding, that position could be targetted for more drastic mutations (for example, a substitution that created in a charge difference) to further reduce binding, if desired.
Alternative methods for obtaining non-cell-binding versions of therapeutic antibodies include genetic techniques such as phage display using error prone PCR (Gram et al, 1992) and cycling of V-region genes (e.g. as sFv constructs) through a bacterial mutator strain (e.g. mutD5) (Low et al., 1996). Such genetic methods can provide powerful screening systems.
The invention will now be further described in the examples which follow. Although the specific example of Campath-1H is given in this document. the invention is not limited to antibodies based on Campath-1H. It is anticipated that other cell-binding therapeutic antibodies, especially those which would be given in repeated doses, will become more widely to accepted using this strategy.